The long term objective of this study is to determine the mechanisms by which changes in intracellular free calcium can regulate growth and cell division. Calcium has been shown to be a universal requirement for cell division. The regulatory mechanisms for controlling the cell division cycle have been shown to be highly conserved. Valuable information concerning the regulation of growth and cell division in humans can be gathered from the study of simpler organisms which lend themselves to genetic manipulation. Growth of one such organism, the filamentous fungus Aspergillus nidulans, has been shown to be dependent upon calcium in the external medium. While much is known about the genes controlling the cell cycle, growth and development in A. nidulans, the role of calcium in these processes is unknown. In order to monitor changes in intracellular free calcium concentrations in vivo, as a function of the cell cycle and during growth and development, recombinant strains of A. nidulas expressing the calcium-indicator protein apoaequorin will be constructed. Three strains in which apoaequorin will be targeted to the nucleus, to mitochondria and to the cytoplasm will be prepared using a strain that exhibits a temperature sensitive defect in the cell cycle. Aequorin will be reconstituted in these strains by incubation in medium containing coelenterazine. Initially, changes in the distribution of intracellular calcium concentrations as a function of cell cycle progression, development, and in response to nitrogen and carbon starvation and refeeding will be measured in cell cultures using a luminometer. Where significant changes in cytoplasmic calcium concentration or localization to the nucleus or mitochondria are found, the distribution of calcium concentrations will be examined using fluorescence microscopy in cells. This will permit the characterization of the type of calcium signal elicited either during a particular stage of the cell cycle or during development or in response to external stimuli such as starvation and refeeding. Characterization of the types of calcium signals should provide information concerning the nature of the signal transduction pathways involved in calcium-mediated processes during cell division, growth, development, and responses to external stimuli.